Che Jinyuan, Hu Shaojie, Fang Qitong, Liu Binghong, Liu Zhuochen, Hu Cunjie, Wang Lei, Li Lekang, Bao Baolong
Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China.
Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China.
J Invertebr Pathol. 2024 Nov;207:108210. doi: 10.1016/j.jip.2024.108210. Epub 2024 Sep 27.
Vibrio parahaemolyticus, a halophilic food-borne pathogen, possesses an arsenal of virulence factors. The pathogenicity of V. parahaemolyticus results from a combination of various virulence factors. HlyA and hlyIII genes are presumed to function in hemolysis, in addition to tdh and trh in V. parahaemolyticus. To confirm the hemolytic function of genes hlyA and hlyIII, ΔhlyA and ΔhlyIII strains of V. parahaemolyticus were separately constructed via homologous recombination. The cytotoxicity and pathogenicity of the ΔhlyA and ΔhlyIII strains were evaluated using a Tetrahymena-Vibrio co-culture model and an immersion challenge in Litopenaeus vannamei. Results indicated that the hemolytic activity of the ΔhlyA and ΔhlyIII strains decreased by approximately 31.4 % and 24.9 % respectively, compared to the WT strain. Both ΔhlyA and ΔhlyIII exhibited reduced cytotoxicity towards Tetrahymena. Then shrimp infection experiments showed LD values for ΔhlyA and ΔhlyIII of 3.06 × 10 CFU/mL and 1.23 × 10 CFU/mL, respectively, both higher than the WT strain's value of 2.57 × 10 CFU/mL. Histopathological observations revealed that hepatopancreas from shrimps challenged with ΔhlyA and ΔhlyIII exhibited mild symptoms, whereas those challenged with the WT strain displayed severe AHPND. These findings indicate that the ΔhlyA and ΔhlyIII strains are significantly less virulent than the WT strain. In conclusion, both hlyA and hlyIII are vital virulence genes involved in hemolytic and cytotoxic of V. parahaemolyticus.
副溶血性弧菌是一种嗜盐性食源性病原体，拥有一系列毒力因子。副溶血性弧菌的致病性是多种毒力因子共同作用的结果。除了副溶血性弧菌中的tdh和trh基因外，HlyA和hlyIII基因被推测在溶血过程中发挥作用。为了证实hlyA和hlyIII基因的溶血功能，通过同源重组分别构建了副溶血性弧菌的ΔhlyA和ΔhlyIII菌株。使用四膜虫 - 弧菌共培养模型和凡纳滨对虾浸泡攻毒实验评估了ΔhlyA和ΔhlyIII菌株的细胞毒性和致病性。结果表明，与野生型菌株相比，ΔhlyA和ΔhlyIII菌株的溶血活性分别降低了约31.4%和24.9%。ΔhlyA和ΔhlyIII对四膜虫的细胞毒性均降低。随后的对虾感染实验表明，ΔhlyA和ΔhlyIII的LD值分别为3.06×10 CFU/mL和1.23×10 CFU/mL，均高于野生型菌株的2.57×10 CFU/mL。组织病理学观察显示，用ΔhlyA和ΔhlyIII攻毒的对虾肝胰腺表现出轻微症状，而用野生型菌株攻毒的对虾则表现出严重的急性肝胰腺坏死病（AHPND）。这些发现表明，ΔhlyA和ΔhlyIII菌株的毒力明显低于野生型菌株。总之，hlyA和hlyIII都是参与副溶血性弧菌溶血和细胞毒性的重要毒力基因。
